System for controlling centrifugal forces to produce cellular monolayers

ABSTRACT

A control system for a slide centrifuge includes a source of light imaged through the slide. A direct light detector and a scattered light detector detect the light. The ratio of the outputs of the two detectors is a signal which is proportional to the spacing of the cells on the slide. The centrifuge motor is stopped when equality occurs between this ratio signal and a decaying signal.

nite States Patent ansfield et a1.

[45] Aug. ,6, 1974 1 SYSTEM FOR CONTROLLING CENTRIFUGAL FORCES TO PRODUCE CELLULAR MONOLAYERS [75] inventors: Gerald R. Mansfield; Charles H. Rogers; Kevin J. Sullivan, all of Raleigh, NC.

[73] Assignee: Corning Glass Works, Corning,

[22] Filed: May 24, 1973 [21] Appl. No: 363,434

[52] US. Cl 356/73, 356/36, 356/197 [5 1] Int. Cl. G0ln 21/00, G0ln 21/24 [58] Field of Search 356/36, 38, 39, 40, 73, 356/103, 196, 197, 201, 206, 212; 350/92;

[56] References Cited UNITED STATES PATENTS 3,572,890 3/1971 Adamik 350/92 Primary ExaminerVincent P. McGraw Attorney, Agent, or Firm-Walter S. Zebrowski; Clarence R. Patty, Jr.

[5 7] ABSTRACT A control system for a slide centrifuge includes a source of light imaged through the slide. A direct light detector and a scattered light detector detect the light. The ratio of the outputs of the two detectors is a signal which is proportional to the spacing of the cells on the slide. The centrifuge motor is stopped when equality occurs between this ratio signal and a decaying signal.

10 Claims, 7 Drawing Figures SCATTERED LIGHT To DIRECT LIGHT RATIO COMPUTATION OUT PAIEIIIEIIIIII: W 3.827.805

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sum 5 nr 5 MOTOR DRIVER INPUT 0 SYSTEM FOR CONTROLLING CENTRIFUGAL FORCES TO PRODUCE CELLULAR MONOLAYERS BACKGROUND OF THE INVENTION This invention relates to control systems for cell smear slide centrifuges, and more particularly to a control system which stops the spinning after an optimum time period which produces a good monolayer of cells.

Examination of cell morphology yields important medical data. Frequently, cell samples are obtained in the form of cells suspended in a liquid. A particular case is the analysis of blood samples. The blood is smeared on a laboratory slide and the smear is stained. By counting the leukocytes on the stained smear, laboratory technicians perform what is referred to as a white blood cell differential. Automation of this differential has significant economic impact because the differential is performed so frequently at every hospital. A thesis by J W. Bacus, An Automated Classification of the Peripheral Blood Leukocytes By Means of Digital Image Processing, University of Illinois, Chicago, 1971, describes one automated system.

Copending application Ser. No. 353,004, filed Apr. 20, 1973, Douglas A. Cotter, Image Scanning Converter For Automated Slide Analysis, describes a system developed by our co-workers for automatically scanning and digitizing the count of the leukocytes on the stained smear.

Spinning the microscope slides wetted with blood is an effective method for preparing a monolayer blood smear which can be automatically processed. Because there is a variation of blood properties, the spin time must be changed in order to produce the best quality blood smears. The copending application of our coworkers, Ser. No. 363,433, filed May 24, 1973, Blood Smeared Slide Centrifuge, discloses a centrifuge in which the spin time is adjusted in accordance with the percent hematocrit of the blood.

US. Pat. No. 3,577,267 Preston et al. and No. 3,705,048 Staunton describe centrifuges which can be used to prepare blood slides but the apparatus described in these patents does not solve the problem of producing blood smears with good cell morphology and good cell distribution for all blood samples.

SUMMARY OF THE INVENTION In accordance with this invention, the spreading apart of the blood cells is monitored as the spin proceeds and the centrifuge motor is stopped at a favorable moment determined by cell spreading and by elapsed time. In this way, the control of the spin is automatically adapted to the blood sample.

In carrying out the invention, a source of light is imaged through the blood-wetted slide. The ratio of the outputs of a direct light detector and a scattered light red cells. For such blood samples, the best compromise is obtained at spin times giving closer packed cells than for low red blood cell content samples. Because of this, the ratio of direct light and scattered light is compared to an exponentially decaying signal. At the time there is an equality between the ratio signal and the exponentially decaying signal, the centrifuge motor is stopped. In this manner, a good monolayer blood smear is obtained without damage to the blood cells which might otherwise be caused by too long a spin time.

This apparatus may be used to make smears from cell suspensions other than blood.

The foregoing and other objects, features and advantages of the invention will be better understood from the following more detailed description and appended claims.

DESCRIPTION OF THE DRAWINGS FIG. 1 shows the basic principle of monitoring the ratio of the direct light to the scattered light;

FIG. 2 is a top view of the platen in the preferred embodiment;

FIG. 3 is a side view of the preferred embodiment;

FIG. 4 depicts a modification of the invention;

FIGS. 5A and 58 together show the circuitry for producing the signal which stops the motor; and

FIG. 6 shows the motor driver circuit.

DESCRIPTION OF THE PREFERRED EMBODIMENT Referring to FIG. 1, light from a source 11 is imaged by the lens 12 onto detector 16 through the slide 13. This slide has a quantity of blood 14 which is centrifuged into a monolayer. The light 15 which is directly transmitted by the slide is imaged upon the direct light detector 16. Early in the spinning operation when the cells are closely spaced, much of the light passing the lens 12 is scattered and constitutes the scattered light 17. The scattered light 17 is detected by the scattered light detector 18. As spinning proceeds and the clear areas between cells enlarge, less light is scattered.

It is desired to produce a signal which represents the spacing between the blood cells. The output of the direct light detector 16 represents the clear area between the cells within the illuminated area of the slide, but this output has other components. For example, the output of the direct light detector 16 is influenced by a dirty optical system, or by the intensity of the light source 1 l. The output of the scattered light detector 18 is representative of the cumulative area of, and therefore the number of, blood cells within the illuminated area of the slide. The output of scattered light detector 18 is also influenced by other components, such as dirt in the optical system or by the intensity of the light source 11. Cancellation of the undesirable signal components is obtained by using the ratio between the direct light detector output and the scattered light detector output to represent the blood cell spacing. The outputs of the two detectors are shown applied to the ratio computation circuit 19 which will subsequently be described in more detail with reference to FIGS. 5A and 5B.

FIGS. 2 and 3 show the preferred arrangement of the detectors and light source in relation to the platen 20. The light from the source 11 is imaged through two holes 21 in the platen. These two holes provide a large area which can be sampled by the light. As the platen spins, two wide arcs centered on the spin center are illuminated. The sampling of a large area such as this prevents small anomalies in the smear from having an unduly large control effect. As the platen rotates, light is intermittently incident upon the detectors l6 and 18. This intermittent action provides a chopper effect and the chopped signals can be precisely processed by the electronics, even if ambient light leaks into the detector, and even if the detector preamplifiers have a DC drift. This chopper action also produces an electrical signal of a frequency determined by the spin speed. This electrical signal is available in the electronics system and can be used for spin speed control if desired.

The centrifuge is mounted in a housing which has a lid 22 for access to the platen. The lid 22 is hinged at 23. The light source 11 is mounted in a recess in the lid and light passes through the pinhole 24 onto the platen. The detectors 16 and 18 are also mounted in the lid. This arrangement provides a convenient and practical mounting for the detectors, light source and lens on the same rigid structure, thereby maintaining a good alignment between them. Also, by mounting the optics on the lid, opening the lid affords easy access for cleaning. In a preferred embodiment, the light source 11 is a continuously visible light emitting diode.

FIG. 4 shows a modification in which light from a collimated source 28 is directed through the hole 29 in the hollow motor shaft 30. A portion of the light beam passes directly through the slide 13 and the blood smear. This light is reflected by beamsplitter 32 to the direct light detector 33. Some of the scattered light passes through the beamsplitter 32 to the scattered light detector 34. Light passing directly through the slide 13, the blood smear, and the beamsplitter 32 is obstructed by the direct light stop 35.

FIGS. A and 5B show the circuitry for stopping the centrifuge motor at the correct time. The photo cells 16 and 18 intercept the direct and scattered light. The signals from these two detectors are amplified by the preamplifiers 36 and 37. Further amplification is provided by tuned AC amplifiers 62 and 63 which are tuned to the frequency at which the light is intermittently incident upon the detectors by reason of the light-chopping action. The amplified signals are rectified in the rectifiers 38 and 39 and are filtered in the low pass filters 40 and 41 to produce a signal D proportional to the instantaneous direct light and a signal S proportional to the instantaneous scattered light. A compensation for the scattered light not due to the blood smear is applied through the resistor 42.

The circuitry produces a signal which stops the centrifuge motor when the ratio D/S equals a decaying threshold. signal. The decaying threshold signal Ae is empirically adjusted to give a good slide appearance for various blood types. /7 Resistor 45 is used to set the time constant T and a scale factor adjustment in the multiplier 46 has the effect of adjusting the factor A. It is a feature of the invention that once having set up the system, no further adjustment is needed, even when widely different blood types are to be smeared. A threshold voltage generator including transistor 43, capacitor 44, and resistor 45 produces the decaying threshold signal. .T he time constant T of the decaying threshold signal is the product of resistance 45 and capacitance 44. The decaying threshold signal is applied to the multiplier 46. The decaying threshold signal is multiplied by the output of the scattered light detector. The product is applied to the comparator 47 where it is compared to the output of the direct light detector. When the two levels become equal,

the comparator changes from the motor run state to the motor stop state. Therefore, the comparator produces a stop signal when The flip-flop 48 is set when the start button 49 is depressed. This applies a positive potential to transistor 52 which starts the centrifuge motor. The inverter 50 provides a 200-millisecond delay interlock. This prevents the light monitor circuit from turning the motor off during the first 200 milliseconds, thereby allowing time for the blood film to form and the sensors to detect the proper light levels.

When the comparator 47 produces a stop signal, the NOR circuit 51 resets the flip-flop 48, thereby stopping the centrifuge motor.

A potentiometer 53 in the emitter circuit of output transistor 52 allows the level of the output to the motor driver to be adjusted to control motor speed.

FIG. 6 shows the motor drive circuit. The input to this circuit comes from the control circuit of FIG. 5. The input is applied to the non-inverting input of amplifier 54 whose output drives the DC motor 55 via the power amplifying transistors 58 and 59. A feedback signal proportional to motor speed and substantially independent of motor current is formed at the inverting input of amplifier 54. Amplifier 54 has a high gain to differential input signals so that the input has to be almost exactly matched by the feedback signal, causing the motor speed to be controlled by the input signal.

The potential from the motor input to ground consists primarily of back EMF which is directly proportional to motor speed and iR drop due to current flow through the various resistive portions of the motor circuit. Motor current flows through resistor 62, generating a potential drop across this resistor directly proportional to the 1R drop in the motor circuit. Amplifier 56 with its two associated resistors produces an output signal directly proportional to the negative of the iR drop in the motor circuit. Resistors 57, 60, and 61 combine the output of amplifier 56 and the potential applied to the motor circuits so that the feedback signal applied to amplifier 54 is independent of motor current and has a convenient scale factor relating motor speed and feedback potential.

The operation is as follows. When the input is switched to a positive potential with the motor initially at rest, transistor 58 supplies a large current to the motor until the motor speed feedback signal equals the input potential. When equality is reached, constant motor speed is maintained. When the input potential is switched to zero, the motor speed feedback signal causes transistor 59 to supply a large reverse current to the motor causing it to slow down and stop. When the motor stops, the feedback signal falls to zero equalling the input signal so that no current is fed to the motor which remains stopped.

Summarizing, when the start button is pushed, the input to the circuit of FIG. 6 goes positive. This turns on transistor 58 to provide a high potential surge that brings the motor 55 up to a desired speed in a short period of time. The back EMF of the motor is used as a feedback signal proportional to actual motor speed. This motor speed signal is applied in a feedback loop which controls the motor spin speed. When the input to the circuit of FIG. 6 returns to the zero level, the transistor 59 is turned on. This applies a reverse polarity current to the motor 55 to bring the motor to a stop in a short period of time.

The system of this invention automatically controls spin time of the centrifuge. In the copending application previously mentioned, Ser. No. 363,433, the spin time is manually changed. It will be appreciated that in many instances it will be desirable to provide both types of control. In one machine which has actually been built, we have incorporated an automatic timing system as disclosed in this invention with a manual override by which the spin time can be manually set.

Within the scope of the invention, many other arrangements of the circuits are possible. Preferred arrangements are those in which the normal tolerances and drifts of the components after initial adjustment cause little change in performance.

While a particular embodiment of the invention has been shown and described, various modifications may be made. The appended claims are therefore intended to cover all such modifications within the true spirit and scope of the invention.

What is claimed is:

l. A control system for a centrifuge for spinning transparent substrates wetted with cell suspensions comprising:

a source of light imaged through the cell suspension wetted transparent substrate,

a direct light detector positioned to detect light transmitted from said source directly through said transparent substrate,

a scattered light detector positioned to detect light from said source which is scattered by the cell suspension covering said transparent substrate,

a centrifuge motor,

means for starting said motor, and

means for stopping said motor when the ratio of the outputs of said direct light detector and said scattered light detector equals a predetermined value.

2. A control system for a centrifuge for spinning transparent substrates wetted with cell suspensions comprising:

a source of light imaged through the cell suspension wetted transparent substrate,

a direct light detector positioned to detect light transmitted from said source directly through said transparent substrate,

a scattered light detector positioned to detect light from said source which is scattered by the cell suspension covering said transparent substrate,

a centrifuge motor,

means for starting said motor,

means for producing a decaying signal which is initiated when said motor is started, and

means for stopping said motor when the ratio of the outputs of said direct light detector and said scattered light detector equals said decaying signal.

3. The system recited in claim 2 wherein said centrifuge includes:

a platen having holes therein, said transparent substrate being positioned on said platen over said holes, said light source being directed through said holes so that light is intermittently incident upon said detectors as said platen rotates.

4. The system recited in claim 3 wherein said direct light detector has a relatively small area disposed in the direct path of light from said source, and wherein said scattered light detector has a larger area than said direct light detector and is positioned to intercept light which is scattered by cell suspension covering said transparent substrate.

5. The system recited in claim 4 further comprising:

a housing for said centrifuge having a lid to provide access to said centrifuge, said light source, said direct light detector and said scattered light detector being mounted on said lid.

6. The system recited in claim 2 further comprising:

an amplifier for the output of said direct light detector tuned to the frequency at which light is intermittently incident upon said detector, and

an amplifier for the output of said scattered light detector tuned to the frequency at which light is intermittently incident upon said scattered light detector.

7. The system recited in claim 2 wherein said means for producing a decaying signal includes:

a resistive capacitive network for generating an exponentially decaying potential,

a multiplier for multiplying said exponentially decaying potential by the output of said scattered light detector,

said means for stopping said motor including a comparator, the output of said direct light detector and the output of said multiplier being applied to said comparator, said comparator producing a signal which stops said motor when the output of said direct light detector equals the product of said scattered light detector and said exponentially decaying potential.

8. The system recited in claim 7 wherein said control system includes:

a variable resistance for adjusting the decay rate of said potential to correspond to the class of cell suspension on the slide.

9. The system recited in claim 2 wherein said centrifuge includes a platen for holding said slide, and

a hollow shaft for driving said platen from said motor, said light source being directed through the hole in said hollow shaft and through said slide to said detectors.

10. The system recited in claim 9 wherein said light source is a collimated light source. 

1. A control system for a centrifuge for spinning transparent substrates wetted with cell suspensions comprising: a source of light imaged through the cell suspension wetted transparent substrate, a direct light detector positioned to detect light transmitted from said source directly through said transparent substrate, a scattered light detector positioned to detect light from said source which is scattered by the cell suspension covering said transparent substrate, a centrifuge motor, means for starting said motor, and means for stopping said motor when the ratio of the outputs of said direct light detector and said scattered light detector equals a predetermined value.
 2. A control system for a centrifuge for spinning transparent substrates wetted with cell suspensions comprising: a source of light imaged through the cell suspension wetted transparent substrate, a direct light detector positioned to detect light transmitted from said source directly through said transparent substrate, a scattered light detector positioned to detect light from said source which is scattered by the cell suspension covering said transparent substrate, a centrifuge motor, means for starting said motor, means for producing a decaying signal which is initiated when said motor is started, and means for stopping said motor when the ratio of the outputs of said direct light detector and said scattered light detector equals said decaying signal.
 3. The system recited in claim 2 wherein said centrifuge includes: a platen having holes therein, said transparent substrate being positioned on said platen over said holes, said light source being directed through said holes so that light is intermittently incident upon said detectors as said platen rotates.
 4. The system recited in claim 3 wherein said direct light detector has a relatively small area disposed in the direct path of light from said source, and wherein said scattered light detector has a larger area than said direct light detector and is positioned to intercept light which is scattered by cell suspension covering said transparent substrate.
 5. The system recited in claim 4 further comprising: a housing for said centrifuge having a lid to provide access to said centrifuge, said light source, said direct light detector and said scattered light detector being mounted on said lid.
 6. The system recited in claim 2 further comprising: an amplifier for the output of said direct light detector tuned to the frequency at which light is intermittently incident upon said detector, and an amplifier for the output of said scattered light detector tuned to the frequency at which light is intermittently incident upon said scattered light detector.
 7. The system recited in claim 2 wherein said means for producing a decaying signal includes: a resistive capacitive network for generating an exponentially decaying potential, a multiplier for multiplying said exponentially decaying potential by the output of said scattered light detector, said means for stopping said motor including a comparator, the output of said direct light detector and the output of said multiplier being applied to said comparator, said comparator producing a signal which stops said motor when the output of said direct light detector equals the product of said scattered light detector and said exponentially decaying potential.
 8. The system recited in claim 7 wherein said control system includes: a variable resistance for adjusting the decay rate of said potential to correspond to the class of cell suspension on the slide.
 9. The system recited in claim 2 wherein said centrifuge includes a platen for holding said slide, and a hollow shaft for driving said platen from said motor, said light source being directed through the hole in said hollow shaft and through said slide to said detectors.
 10. The system recited in claim 9 wherein said light source is a collimated light soUrce. 